Hydroxyapatite nanoparticles were isolated from a biowaste, buffalo bone, via the thermal decomposition method. The resulting white powdered material was characterized by Fourier Transformed Infrared (FTIR) spectroscopy, X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), and Energy Dispersive X-ray (EDX) analysis. The FTIR spectra confirmed that a heat treatment of the bone powder at the temperature at or above 1223 K removed the organic moieties leading to the formation of a pure inorganic biomineral. The XRD analyses showed that the obtained material was nanocrystalline HAp (nano-HAp) with an average grain diameter of 25 nm, while their rod-shaped particles with their tightly agglomerated morphology were confirmed by the SEM analysis. Besides Calcium (Ca), Phosphorous (P), and Oxygen (O), trace amounts of Aluminum (Al), Magnesium (Mg), Copper (Cu), Zirconium (Zr) and Carbon (C) were also found by EDX analysis. Antibacterial activity of nano-HAp against six standard isolates was investigated by the agar well diffusion method and found to be more susceptible to Acinetobacter baumannii while other standard strains such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus showed lesser susceptibility and no antibacterial activity was noticed against Salmonella typhi and Methicillin resistant Staphylococcus aureus (MRSA) with the analysed concentration of nano-HAp suggesting its potential application in biomedical fields.